赤小豆加工食品中红豆成分定性和定量PCR检测

Qualitative and quantitative detection of adzuki bean ingredient in rice bean processed foods

目的为实现赤小豆加工食品的真伪和品质鉴别,建立赤小豆加工食品中被误用或混用的红豆成分实时荧光聚合酶链式反应(PCR)定性和微滴数字聚合酶链式反应(ddPCR)定量检测方法。方法根据赤小豆和红豆基因组DNA中保守基因,分别设计适用于赤小豆和红豆成分实时荧光PCR定性检测的特异性引物探针,并设计适用于赤小豆加工食品中红豆成分双重ddPCR定量检测的通用引物探针,建立赤小豆加工食品中红豆成分质量百分比-DNA拷贝数浓度百分比线性关系式。结果本方法对赤小豆和红豆基因组DNA检测低限分别为0.1和0.01 ng/μL,对赤小豆和红豆基因组DNA拷贝数浓度定量检测限均为6 copies/μL,可对赤小豆加工食品中质量百分比为5%~80%的红豆成分准确定量。结论本方法可用于赤小豆加工食品中红豆成分的定性和质量百分比定量检测。

Objective In order to achieve the authenticity and quality identification of rice bean processed food,qualitative real-time fluorescent polymerase chain reaction(PCR)and quantitive droplet digital PCR(ddPCR)methods were established for adzuki bean ingredients misused or mixed in rice bean processed food.Methods Specific primers and probes for qualitative real-time fluorescent PCR were designed according to their conserved sequences in genomic DNA,as well as universal primers and probe for quantification of adzuki bean ingredient in rice bean processed foods using duplex droplet digital PCR.Then a linear formula of mass ratio-DNA copy was established.Results The LODs of realtime fluorescent PCR for rice bean and adzuki bean were 0.1 and 0.01 ng/μL separately,and the LOQs of ddPCR for both were 6 copies/μL.Accurate quantification of adzuki bean ingredient with a mass ratio from 5%to 80%in rice bean processed foods therefore could be achieved.Conclusion This method could be used for qualitative and mass ratio quantification determination of adzuki bean ingredients in rice bean processed food.